The main mechanisms of action for therapeutic IgG antibodies are direct effects (e.g., apoptosis), ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). The human IgG1 isotype in particular can induce strong ADCC and CDC when compared with the other heavy chain isotypes. CDC effector functions are activated through the interactions of the fragment crystallizable (“Fc”) regions of IgG molecules with complement. An Fc region of an immunoglobulin is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains (depicted in FIG. 1).
CDC is a cytolytic cascade mediated by a series of complement proteins present in serum. It is triggered by the binding of complement protein C1q to the Fc region of an antibody molecule (Natsume et al., 2009, Drug Des. Devel. Ther. 3:7-16). The signaling pathway of the complement system, shown in FIG. 2, triggers targeted cellular destruction via the membrane-attack complex (MAC), a transmembrane channel formed by association of complement proteins. Loss of membrane integrity via the MAC results in cell swelling and lysis.
Antibodies with enhanced CDC are thought to be more effective as therapeutics. Arzerra (ofatumumab) is an anti-CD20 antibody used in the treatment of chronic lymphocytic leukemia (CLL). Ofatumumab was selected on the basis of its higher CDC activity compared to an earlier anti-CD20 antibody, Rituxan (Pawluczkowycz et al., 2009, J. Immunol. 183:749-758). CDC is also thought to be an important mechanism of action of the anti-CD52 antibody alemtuzumab (Campath-1H) (Natsume et al., 2009, Drug Des. Devel. Ther. 3:7-16).
The binding of the C1q component to the Fc, the initial step of the complement cascade, affects the intensity of the following complement activation, and several approaches have succeeded in enhancing CDC by facilitating the binding of the antibody constant region to C1q. As a result of engineered amino acid mutations inserted into either Fc or the hinge region, designed antibody constant regions possessing improved C1q binding have been achieved (Natsume et al., 2009, Drug Des. Devel. Ther. 3:7-16).
Nonetheless, there remains a need for identification of further amino acid substitutions in Fc molecules that modulate the interaction between Fc and C1q in order to enhance CDC activation and therefore efficacy of Fc-containing therapeutic molecules, particularly for indications such as cancer where cell death of the target cell is desired.